As known, eucalyptus is widely planted in Asia, South America and some parts of Europe, especially in the Mediterranean region. This kind offers a range of advantages over other forest species for use in the cellulose industry, such as quick growing, regeneration from multiple sprouting after harvesting and capacity to grow in poor soils. Various research groups in many countries, including public and private institutions have attempted genetic transformation of eucalyptus. Few reports have however shown stable gene transference for different eucalyptus species.
Serrano et al., in their article “Genetic transformation of Eucalyptus globulus through biollistics: complementary development of procedures for organogenesis form zygotic embryos and stable transformation of corresponding proliferation tissue”, J. Exp. Botany 47 (295), 1996, 285–290, discloses a particle bombing (bioballistic) method to introduce DNA molecules containing gene uidA coding the β-glucuronidase (GUS) protein into zygotic embryos of Eucalyptus globulus. After two months of bombing, callus were obtained which presented positive GUS reaction. Although tDNA integration was shown by hybridization through the Southern Blot method, plants were not regenerated.
Moralejo et al., “Generation of transgenic Eucalyptus globulus plantlets through Agrobacterium tumefaciens mediated transformation”, J. Plant Physiol., 25:297–212, 1998, obtained transgenic E. globulus plants by making use of the gene transference method via Agrobacterium tumefaciens, infecting plantlets which had been injured with micro-particles bombing before the co-culture. However, the efficiency in obtaining transgenic plants was very low, around 1%.
Mullins et al., “Regeneration and transformation of Eucalyptus camaldulensis”, Plant Cell Reports, 16, 1997, 787–791, obtained plant regeneration from sprouts regenerated from foliar explants and transformation of a clone of E. camaldulensis, by making use of the transformation system as mediated by Agrobacterium tumefaciens. 
Ho et al., “Agrobacterium tumefaciens—mediated transformation of Eucalyptus camaldulensis and production of transgenic plants”, Plant Cell Rep., 17, 1998, 675–680, also described the transformation and regeneration of E. camaldulensis, by making use of the Agrobacterium system to transform hypocotyls. In all these examples, low transformation efficiency was the main limiting factor to the large-scale use of such methods to introduce exogenous genes in eucalyptus.
Various methods have been used to optimize the efficiency of eucalyptus transformation by making use of the Agrobacterium system. Recently, a transformation system was developed using ultrasound and sonication to increase the efficiency of Agrobacterium penetration within target tissues for transformation. The method called SAAT (“Sonication Assisted Agrobacterium Transformation”) was used to increase the efficiency of gene transference in soy, peas, wheat and corn (Trick & Finer, “SAAT-sonication assisted Agrobacterium mediated transformation”, Transg. Res., 6:329–336, 1997, “Sonicated assisted Agrobacterium mediated transformation of soybean [Glycine max (L.) Merrill] embryogenic suspension culture tissue”, Plant Cell Rep., 17:482–488, 1998, Santarém et al., “Sonication assisted Agrobacterium mediated transformation of soybean immature cotyledons: optimization of transient expression”, Plant Cell Rep., 17:752–759, 1998).
The use of ultrasound for plant tissues induces acoustic cavitation, generating microscopic injuries, which are channels to favor the internal exposure of tissues to Agrobacterium (Joersbo & Brunstedt, “Sonication: A new method for gene transfer to plants”, Physiol. Plant, 85:230–234, 1992), thus increasing tDNA transitory expression levels. For eucalyptus, most tissues used for the genetic transformation are cotyledons, hypocotyls, immature embryos and callus obtained from different plant tissues.
A bioballistic or biollistic method is also known, consisting in firing micro-projectiles of gold or platinum containing precipitated DNA on their surfaces. The projectile goes through the cellular wall/membrane carrying DNA with it. To launch projectiles, equipment similar to a pressure rifle is used, in which pressure is produced by an inert gas. Such procedure is used for the genetic transformation of plants and animals.
Still on this issue, for chemical analysis procedures, ultrasonic wave generator systems are employed to prepare samples, be it in processes to extract chemical species or be it to dissolve solid samples. The base for ultrasonic application to such processes is related to the shock waves resulting from the application of an acoustic field over a material means. Such waves increase the interaction between the solvent and the surface of the solids, increasing the concentration of species present in the material being investigated in the solution. The vibration caused by ultrasounds minimizes the concentration gradient in the surroundings of the surface of the solid present in the exposed medium and makes possible the transport of salts and oxides from the surface of the solid to the solution. When ultrasound probes are employed, however, the association of magnetic agitators to the system should not be disregarded, as only a small zone of the solution, in which the probe is immersed, will be submitted to the high intensity produced by such processors and vigorous stirring provides the same level of interaction for the whole exposed solution.
In the sonication method, rupture of cells is obtained, thus allowing for gene introduction for the final genetic transformation. The use of sonication aims at increasing the transformation efficiency, since it causes injuries to the tissues, which then release phenol compounds, increasing the attraction of bacteria to those regions and facilitating gene transference.
The previously used bioballistic method for Eucalyptus did not encompass regeneration with β-glucuronidase (GUS) to the recovery of the final plant.
A method to use Agrobacterium with final regeneration into E. camaldulensis under low efficiency has also been reported.
Patent application WO 9625504-dated Aug. 22, 1996 describes a process to obtain transgenic plants by means of incorporation of a stable DNA sequence of interest. The process comprises cells or tissues of Eucalyptus with Agrobacterium being the mediator for the DNA sequence transference, inducing the formation of callus with phenyl urea derivatives in the presence of geneticin (G-418). The process is useful to transform clone material directly or indirectly derived from a mature Eucalyptus tree. 